Adenovirus carrying gag gene HIV vaccine

ABSTRACT

An adenoviral vector is described which carries a codon-optimized gag gene, along with a heterologous promoter and transcription terminator. This viral vaccine can effectively prevent HIV infection when administered to humans either alone or as part of a prime and boost regime also with a vaccine plasmid.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of PCT InternationalApplication No. PCT/US00/18332, filed Jul. 3, 2000, which designates theU.S., which claims the benefit, under 35 U.S.C. §119(e), of U.S.Provisional Application Serial No. 60/148,981, filed Aug. 13, 1999 andU.S. Provisional Application Serial No. 60/142,631, filed Jul. 6, 1999.

FIELD OF THE INVENTION

[0002] This invention relates to replication deficient adenovirusvectors comprising an optimized human immunodeficiency virus (HIV) gaggene under the control of a strong promoter, which are suitable forvaccines against HIV.

BACKGROUND OF THE INVENTION

[0003] Human Immunodeficiency Virus-1 (HIV-1) is the etiological agentof acquired human immune deficiency syndrome (AIDS) and relateddisorders.

[0004] Vaccination is an effective form of disease prevention and hasproven successful against several types of viral infection. However,determining ways to present HIV-1 antigens to the human immune system inorder to evoke protective humoral and cellular immunity is a difficulttask. In AIDS patients, free virus is present in low levels only.Transmission of HIV-1 is enhanced by cell-to-cell interaction via fusionand syncytia formation. Hence, antibodies generated against free virusor viral subunits are generally ineffective in eliminatingvirus-infected cells.

[0005] European Patent Applications 0 638 316 (Published Feb. 15, 1995)and 0 586 076 (Published Mar. 9, 1994), (both assigned to American HomeProducts Corporation) describe replicating adenovirus vectors carryingan HIV gene, including env or gag. Various treatment regimens were usedwith chimpanzees and dogs, some of which included booster adenovirus orprotein plus alum treatments.

[0006] Infection with HIV-1 is almost always fatal, and at present thereare no cures for HIV-1 infection. Effective vaccines for prevention ofHIV-1 infection are not yet available. Because of the danger ofreversion or infection, live attenuated virus probably cannot be used asa vaccine, and. subunit vaccine approaches have not been successful atpreventing HIV infection. Treatments for HIV-1 infection, whileprolonging the lives of some infected persons, have serious sideeffects. There is thus a great need for effective treatments andvaccines to combat this lethal infection.

SUMMARY OF THE INVENTION

[0007] This invention relates to a vaccine composition comprising areplication- defective adenoviral vector comprising at least one geneencoding an HIV gag protein, wherein the gene comprises codons optimizedfor expression in a human, and the gene is operably linked to aheterologous promoter.

[0008] Another aspect of this invention relates to an adenoviral vaccinevector comprising: a replication defective adenoviral genome, whereinthe adenoviral genome does not have a functional E1 gene, and theadenoviral genome further comprises a gene expression cassettecomprising:

[0009] i) a nucleic acid encoding a HIV gag protein, wherein the nucleicacid is codon optimized for expression in a human host;

[0010] ii) a heterologous promoter is operatively linked to the nucleicacid encoding the gag protein; and

[0011] iii) a transcription terminator.

[0012] In preferred embodiments, the E1 gene has been deleted from theadenoviral vector, and the HIV expression cassette has replaced thedeleted E1 gene. In other preferred embodiments, the replicationdefective adenovirus genome does not have a functional E3 gene, andpreferably the E3 gene has been deleted.

[0013] This invention also relates to a shuttle plasmid vectorcomprising: an adenoviral portion and a plasmid portion, wherein saidadenovirus portion comprises: a) a replication defective adenovirusgenome which does not have a functional E1 gene; and b) a geneexpression cassette comprising: a nucleic acid encoding an HIV gagprotein, wherein the nucleic acid is codon optimized for expression in ahuman host; a heterologous promoter operably linked to the nucleic acidencoding the gag protein; and a transcription terminator.

[0014] Other aspects of this invention include a host cell comprisingthe adenoviral vaccine vectors and/or the shuttle plasmid vectors,methods of producing the vectors comprising introducing the adenoviralvaccine vector into a host cell which expresses adenoviral E1 protein,and harvesting the resultant adenoviral vaccine vectors.

[0015] Another aspect of this invention is a method of generating acellular immune response against an HIV protein in an individualcomprising administering to the individual an adenovirus vaccine vectorcomprising:

[0016] a) a replication defective adenoviral vector, wherein theadenoviral vector does not have a functional E1 gene, and

[0017] b) a gene expression cassette comprising: i) a nucleic acidencoding an HIV gag protein, wherein the nucleic acid is codon optimizedfor expression in a human host; ii) a heterologous promoter operativelylinked to the nucleic acid encoding the gag protein; and iii) atranscription terminator.

[0018] In some embodiments of this invention, the individual is givenmore than one administration of adenovirus vaccine vector, and it may begiven in a regiment accompanied by the administration of a plasmidvaccine. The plasmid vaccine comprises a plasmid encoding acodon-optimized gag protein, a heterologous promoter operably linked tothe gag protein nucleic acids, and a transcription terminator. There maybe a predetermined minimum amount of time separating theadministrations. The individual can be given a first dose of plasmidvaccine, and then a second dose of plasmid vaccine. Alternatively, theindividual may be given a first dose of adenovirus vaccine vector, andthen a second dose of adenoviral vaccine vector. In other embodiments,the plasmid vaccine is administered first, followed after a time byadministration of the adenovirus vector vaccine. Conversely, theadenovirus vaccine vector may be administered first, followed byadministration of plasmid vaccine after a time. In these embodiments, anindividual may be given multiple doses of the same adenovirus serotypein either viral vector or plasmid form, or the virus may be of differingserotypes.

BRIEF DESCRIPTION OF THE FIGURES

[0019]FIG. 1 is a graph showing the number of gag peptide-specificinterferon-gamma secreting splenocytes (×10⁶) from rats which wereimmunized with gag plasmid or Ad5FLgag.

[0020]FIG. 2 shows serum SEAP (secreted alkaline phosphatase) expressionlevels in rhesus monkeys following injection with FG Ad5-SEAP or SEAPDNA constructs.

[0021]FIGS. 3A, 3B and 3C show anti-HIV gag cytotoxic T lymphocyteresponses in three rhesus monkeys vaccinated with FG Ad5 tPAgag. Eachpanel represents the specific killing response of a particular monkeys(denoted as numbers 92×024 in FIG. 3A, 94×012 in FIG. 3B, and 94×025 inFIG. 3C) at various time points following immunization at 0, 8, and 24weeks. The abscissa axis shows the effector/target (E/T) ratios ofcultured T cells and B cells employed in this assay, while the ordinateaxis shows specific lysis values obtained for each sample. Specificlysis values of at least 10% difference between curves±gag peptideantigen are generally considered significant. The square symbolsrepresent target cells treated with an irrelevant influenza peptideantigen while the circles, triangles, and diamonds represent targetcells treated with partial or complete gag peptide pools, respectively.

[0022] FIGS. 4A-H show anti-HIV gag cytotoxic T lymphocyte responses inrhesus monkeys vaccinated with FG Ad5FLgag. FIGS. 4A, B, and C are thefirst group of monkeys, D, E, and F are the second, and G, H, and I arethe third group. Each represents specific killing responses of eachmonkey receiving the indicated treatment. The abscissa axis shows theeffector/target (E/T) ratios of cultured T cells and B cells employed inthis assay, while the ordinate axis shows specific lysis values obtainedfor each sample. Specific lysis values of at least 10% differencebetween curves±gag peptide antigen are generally considered significant.The square symbols represent target cells treated with DMSO alone at thesame concentration as samples containing peptides while the circles,triangles, and diamonds represent target cells treated with partial (F,G) or complete (H) gag peptide pools, respectively.

[0023] FIGS. 5A-H show anti-HIV gag cytotoxic T lymphocyte responses inrhesus monkeys vaccinated with Ad2Flgag priming, followed by eitherAd2Flgag or Ad5Flgag boosting. Each panel (FIGS. 5A-G) representsspecific killing responses of a group of three monkeys receiving theindicated treatment. The last panel (FIG. 5H) shows responses from twonaive monkeys that were not vaccinated. The abscissa axis shows theeffector/target (E/T) ratios of cultured T cells and B cells employed inthis assay, while the ordinate axis shows specific lysis values obtainedfor each sample. Specific lysis values of at least 10% differencebetween curves±gag peptide antigen are generally considered significant.

[0024]FIG. 6 is the nucleic acid sequence (SEQ.ID.NO.1) of the optimizedhuman HIV-1 gag open reading frame.

[0025]FIG. 7A shows construction of the adenovirus carryingcodon-optimized gag. FIG. 7B shows construction of the adenoviruscarrying codon-optimized tPA-gag.

[0026]FIG. 8 is the nucleic acid sequence of the optimized tPA-gag openreading frame.

[0027]FIG. 9 show the longevity (69 weeks after final boost) of cellularimmune responses in rhesus monkeys immunized with Ad5FLgag

[0028]FIG. 10 shows CMI repsonses prior to and subsequent to a week 24boost with Ad5-FLgag (SFC/10⁶ cells via ELIspot)

[0029]FIG. 11 shows long term CMI responses for an HIV gag DNA vaccine(0, 4 and 8 weeks) and Ad5FLgag (single prime at T=0) which were boostedwith 10⁷ particles of Ad5FLgag (SFC/10⁶ cells via ELIspot).

[0030]FIG. 12 shows a comparison of various single modality Ad5FLgagimmunizations with DNA gag vaccines adjuvanted with various formulationsof CRL1005. (D113:5 mg/ml DNA, 7.5 mg CRL1005 in PBS; D117: 5 mg/ml DNA,22.5 mg CRL1005 in PBS; D118: 7.5 mg/mL CRL1005, 0.5 mM BAK and 5 mg/mLDNA in PBS. DNA/POP-POE/BAK formulations are disclosed in U.S.provisional application Ser. Nos. 60/214,824 and 60/213622, filed Jun.28, 2000 and Jun. 23, 2000, respectively; both of which are herebyincorporated by reference.) Four columns of data are presented for eachanimal. The far left column is a pre-immunization ELISPOT response; thecolumn second from the left represents the ELISPOT after either the Ad5or DNA priming, respectively; the column third from the left is takenthe week of or prior to the boosting, and the fourth column measures aCMI response subsequent to the Ad5FLgag boost.

[0031] FIGS. 13A-E shows the longitudinal p11C-specific tetramerstaining results for all Mamu-A*01 monkeys up to one week beforechallenge. These data are presented as a percentage of the CD3⁺CD8⁺positive T cell population. Arrows indicate time of innoculations. (A)SIV gag DNA; (B) MVA-SIV gag; (C) SIV gag DNA+alum/MPL; (D) Ad5-SIV gag;and (E) SIV gag DNA+CRL1005.

[0032] FIGS. 14A-F show post challenge longitudinal results forperipheral p11C-specific tetramer staining for each group, as follows:(A) SIV gag DNA; (B) SIV gag DNA+CRL1005; (C) SIV gag DNA+alum/MPL; (D)MVA-SIV gag; (E) Ad5-SIV gag; and, (F) naïve animals.

[0033] FIGS. 15A-L shows post-challenge CD4 T cell counts (A, C, E, G,and I) and plasma viral load (B, D, F. H, J, L) for each group, asfollows: (A, B) SIV gag DNA; (C,D) SIV gag DNA+CRL1005; (E, F) SIV gagDNA+alum/MPL; (G, H) MVA-SIV gag; (I, J) Ad5-SIV gag; and, (K, L) naïveanimals.

[0034] As used throughout the specification and claims, the followingdefinitions and abbreviations are used:

[0035] In general, adenoviral constructs, gene constructs are named byreference to the genes contained therein, such as below:

[0036] “tPAgag” refers to a fusion between the leader sequence of thetissue plasminogen activator leader sequence and an optimized HIV gaggene.

[0037] “Ad5-tPAgag” refers to an adenovirus serotype 5 replicationdeficient virus which carries an expression cassette which comprises atissue plasminogen activator leader sequence fused to a codon-optimizedgag gene which is under the control of the CMV promoter and containsIntron A.

[0038] “Fl” refers to a full length gene.

[0039] “Flgag” refers to the full-length optimized gag gene.

[0040] “Ad5-Flgag” refers to an adenovirus serotype 5 replicationdeficient virus which carries an expression cassette which comprises afull length optimized gag gene under the control of the CMV promoter andcontains Intron A.

[0041] “FG Adenovirus” means a First Generation adenovirus, i.e. areplication deficient adenovirus which has either a non-functional ordeleted E1 region, and optionally a non-functional or deleted E3 region.

[0042] “Promoter” means a recognition site on a DNA strand to which anRNA polymerase binds. The promoter forms an initiation complex with RNApolymerase to initiate and drive transcriptional activity. The complexcan be modified by activating sequences such as enhancers or inhibitingsequences such as silencers.

[0043] “Leader” means a DNA sequence at the 5′ end of a structural genewhich is transcribed along with the gene. This usually results a proteinhaving an N-terminal peptide extension, often referred to as apro-sequences.

[0044] “Intron” as used herein refers to a section of DNA occurring inthe middle of a gene which does not code for an amino acid in the geneproduct. The precursor RNA of the intron is excised and is therefore nottranscribed into mRNA not translated into protein.

[0045] “Cassette” refers to the a nucleic acid sequence which is to beexpressed, along with its transcription and translational controlsequences. By changing the cassette, a vector can express a differentsequence.

[0046] It has been found according to this invention that firstgeneration adenoviral vectors carrying a codon-optimized HIV gag generegulated with a strong heterologous promoter can be used as humananti-HIV vaccines, and are capable of inducing immune responses.

[0047] The adenoviral vector which makes up the backbone of the vaccineconstruct of this invention is preferably a “first generation”adenoviral vector. This group of adenoviral vectors is known in the art,and these viruses are characterized by being replication-defective. Theytypically have a deleted or inactivated E1 gene region, and preferablyadditionally have a deleted or inactivated E3 gene region. In apreferred embodiment of this invention, the first generation replicationincompetent adenovirus vector used is a serotype 5 adenovirus containingdeletions in E1 (Ad5 base pairs 342-3523) and E3 (Ad5 base pairs 28133to 30818). For adenovirus 2 serotype, the E1 deletions are preferably bp559-3503 and the E3 deletions are preferably 28,812-29,773. (Genbankgb:J01917). Those of skill in the art can easily determine theequivalent sequences for other serotypes, such as serotypes 4, 12, 6,17, 24, 33, 42, 31, 16.

[0048] Adenoviral serotypes 2 and 5, particularly 5 are preferred foruse in this invention, since at this point in time, more is known aboutthese serotypes generally than other serotypes, and their complete DNAsequences are known. The prototype serotype 5 adenovirus has beencompletely sequenced (Chroboczek et al, 1992 J. Virology 186:280, whichis hereby incorporated by reference.) They also belong to the subgroup Cadenoviruses, which are not associated with human or rodentmalignancies. However, it is envisioned that any adenovirus serotype canbe used in this invention, including non-human ones, as deletion of E1genes should render all adenoviruses non-tumorogenic. Also it may beadvantageous to use a serotype which has less prevalence in the wild, aspatients are less likely to have previous exposure (and lesspre-existing antibodies) to a rarer serotype.

[0049] The adenoviral vectors can be constructed using known techniques,such as those reviewed in Hitt et al, 1997 “Human Adenovirus Vectors forGene Transfer into Mammalian Cells” Advances in Pharmacology 40:137-206,which is hereby incorporated by reference.

[0050] In constructing the adenoviral vectors of this invention, it isoften convenient to insert them in to a plasmid or shuttle vector. Thesetechniques are known and described in Hitt et al supra. This inventionspecifically includes both the adenovirus and the adenovirus wheninserted into a shuttle plasmid.

[0051] Viral vectors can be propagated in various E1 complementing celllines, including the known cell lines 293 and PER.C6. Both these celllines express the adenoviral E1 gene product. PER.C6 is described in WO97/00326, published Jan. 3, 1997, which is hereby incorporated byreference. It is a primary human retinoblast cell line transduced withan E1 gene segment that complements the production of replicationdeficient (FG) adenovirus, but is designed to prevent generation ofreplication competent adenovirus by homologous recombination. 293 cellsare described in Graham et al 1977 J. Gen. Virol 36:59-72, which ishereby incorporated by reference.

[0052] The HIV gag gene selected to be expressed is of importance to theinvention. Sequences for many genes of many HIV strains are publiclyavailable in GENBANK and primary, field isolates of HIV are availablefrom the National Institute of Allergy and Infectious Diseases (NIAID)which has contracted with Quality Biological (Gaithersburg, Md.) to makethese strains available. Strains are also available from the WorldHealth Organization (WHO), Geneva Switzerland. In a preferred embodimentof this invention, the gag gene is from an HIV-1 strain (CAM-1; Myers etal, eds. “Human Retroviruses and AIDS: 1995, IIA3-IIA19, which isincorporated by reference). This gene closely resembles the consensusamino acid sequence for the clade B (North American/European) sequence.

[0053] Regardless of the HIV gene chosen for expression, the sequenceshould be “optimized” for expression in a human cellular environment. A“triplet” codon of four possible nucleotide bases can exist in 64variant forms. That these forms provide the message for only 20different amino acids (as well as transcription initiation andtermination) means that some amino acids can be coded for by more thanone codon. Indeed, some amino acids have as many as six “redundant”,alternative codons while some others have a single, required codon. Forreasons not completely understood, alternative codons are not at alluniformly present in the endogenous DNA of differing types of cells andthere appears to exist variable natural hierarchy or “preference” forcertain codons in certain types of cells. As one example, the amino acidleucine is specified by any of six DNA codons including CTA, CTC, CTG,CTT, TTA, and TTG (which correspond, respectively, to the mRNA codons,CUA, CUC, CUG, CUU, UUA and UUG). Exhaustive analysis of genome codonfrequencies for microorganisms has revealed endogenous DNA of E. colimost commonly contains the CTG leucine-specifying codon, while the DNAof yeasts and slime molds most commonly includes a TTAleucine-specifying codon. In view of this hierarchy, it is generallyheld that the likelihood of obtaining high levels of expression of aleucine-rich polypeptide by an E. coli host will depend to some extenton the frequency of codon use. For example, a gene rich in TTA codonswill in all probability be poorly expressed in E. coli, whereas a CTGrich gene will probably highly express the polypeptide. Similarly, whenyeast cells are the projected transformation host cells for expressionof a leucine-rich polypeptide, a preferred codon for use in an insertedDNA would be TTA.

[0054] The implications of codon preference phenomena on recombinant DNAtechniques are manifest, and the phenomenon may serve to explain manyprior failures to achieve high expression levels of exogenous genes insuccessfully transformed host organisms—a less “preferred” codon may berepeatedly present in the inserted gene and the host cell machinery forexpression may not operate as efficiently. This phenomenon suggests thatsynthetic genes which have been designed to include a projected hostcell's preferred codons provide a preferred form of foreign geneticmaterial for practice of recombinant DNA techniques. Thus, one aspect ofthis invention is an adenovirus vector which specifically includes a gaggene which is codon optimized for expression in a human cellularenvironment.

[0055] The diversity of function that typifies eukaryotic cells dependsupon the structural differentiation of their membrane boundaries. Togenerate and maintain these structures, proteins must be transportedfrom their site of synthesis in the endoplasmic reticulum topredetermined destinations throughout the cell. This requires that thetrafficking proteins display sorting signals that are recognized by themolecular machinery responsible for route selection located at theaccess points to the main trafficking pathways. Sorting decisions formost proteins need to be made only once as they traverse theirbiosynthetic pathways since their final destination, the cellularlocation at which they perform their function, becomes their permanentresidence.

[0056] Maintenance of intracellular integrity depends in part on theselective sorting and accurate transport of proteins to their correctdestinations. Over the past few years the dissection of the molecularmachinery for targeting and localization of proteins has been studiedvigorously. Defined sequence motifs have been identified on proteinswhich can act as “address labels”. Leader or signal peptides such asthat from the tissue-specific plasminogen activator protein, tPA, serveto transport a protein into the cellular secretory pathway through theendoplasmic reticulum and golgi apparatus. A number of sorting signalshave been found associated with the cytoplasmic domains of membraneproteins such as di-Leucine amino acid motifs or tyrosine-basedsequences that can direct proteins to lysosomal compartments. For HIV,transport and extrusion from the cell of viral particles depend uponmyristoylation of glycine residue number two at the amino terminus ofgag. In some embodiments of the optimized gag gene, the tPA leadersequence has been attached 5′ to the structural gene sequence.

[0057] The optimized gag gene is incorporated into an expressioncassette. The cassette contains a transcriptional promoter recognized byan eukaryotic RNA polymerase, and a transcriptional terminator at theend of the gag gene coding sequence. In a preferred embodiment, thepromoter is a “strong” or “efficient” promoter. An example of a strongpromoter is the immediate early human cytomegalovirus promoter (Chapmanet al, 1991 Nucl. Acids Resl9:3979-3986, which is incorporated byreference) with the intron A sequence (CMV-intA), although those skilledin the art will recognize that any of a number of other known promoters,such as the strong immunoglobulin, or other eukaryotic gene promotersmay be used, including the EF1 alpha promoter, the murine CMV promoter,Rous sarcoma virus (RSV) promoter, SV40 early/late promoters and thebeta-actin promoter. A preferred transcriptional terminator is thebovine growth hormone terminator. The combination of CMVintA-BGHterminator is particularly preferred although other promoter/terminatorcombinations in the context of FG adenovirus may also be used.

[0058] To assist in preparation of the polynucleotides in prokaryoticcells, a shuttle vector version of the adenovirus vector is oftenprepared. The shuttle vector contains an adenoviral portion and aplasmid portion. The adenoviral portion is essentially the same as theadenovirus vector discussed supra, containing adenoviral sequences (withnon-functional or deleted E1 and E3 regions) and the gag expressioncassette, flanked by convenient restriction sites. The plasmid portionof the shuttle vector often contains an antibiotic resistance markerunder transcriptional control of a prokaryotic promoter so thatexpression of the antibiotic does not occur in eukaryotic cells.Ampicillin resistance genes, neomycin resistance genes and otherpharmaceutically acceptable antibiotic resistance markers may be used.To aid in the high level production of the polynucleotide byfermentation in prokaryotic organisms, it is advantageous for theshuttle vector to contain a prokaryotic origin of replication and be ofhigh copy number. A number of commercially available prokaryotic cloningvectors provide these benefits. It is desirable to remove non-essentialDNA sequences. It is also desirable that the vectors not be able toreplicate in eukaryotic cells. This minimizes the risk of integration ofpolynucleotide vaccine sequences into the recipients' genome.Tissue-specific promoters or enhancers may be used whenever it isdesirable to limit expression of the polynucleotide to a particulartissue type.

[0059] In one embodiment of this invention, the shuttle plasmid used ispAD.CMVI-FLHIVgag, was made using homologous recombination techniques.For clinical use, the shuttle vector was rescued into virus in PER.C6cells. To rescue, the shuttle plasmid was linearized by PacI restrictionenzyme digestion and transfected into the PER.C6 cells using the calciumphosphate coprecipitate method. The plasmid in linear form is capable ofreplication after entering the PER.C6 cells and virus is produced. Theinfected cells and media were harvested after viral replication wascomplete.

[0060] Standard techniques of molecular biology for preparing andpurifying DNA constructs enable the preparation of the DNA immunogens ofthis invention.

[0061] To ensure a clonal virus population a method of clonalpurification was used for clinical material. The virus obtained fromtransfection of the PER.C6 cells was serially diluted to extinctionusing 2-fold dilutions. The dilutions were then used to infect PER.C6cells in 96 well plates using 24 wells for each solution At the end of a14-day incubation period the wells were scored positive or negativeusing adenovirus specific PCR and gag ELISA. Virus positive wells at thehighest dilutions were selected for expansion. The selected well was theonly positive well out of 24 wells plated at that dilution giving 98%assurance of clonality Verification of that endpoint had been reached inthe dilution series, and that virus positive wells that had insufficientvirus to be detected in the initial screening had not been missed, wasobtained by subculturing the original 96 well plated two additionaltimes and rescoring them This confirmed the clonality of the selectedwell. The selected virus was designated AD5FLgag.

[0062] The adenoviral vaccine composition may contain physiologicallyacceptable components, such as buffer, normal saline or phosphatebuffered saline, sucrose, other salts and polysorbate. One preferredformulation has: 2.5-10 mM TRIS buffer, preferably about 5 mM TRISbuffer; 25-100 mM NaCl, preferably about 75 mM NaCl; 2.5-10% sucrose,preferably about 5% sucrose; 0.01 -2 mM MgCl₂; and 0.001%-0.01%polysorbate 80 (plant derived). The pH should range from about 7.0-9.0,preferably about 8.0. One skilled in the art will appreciate that otherconventional vaccine excipients may also be used it make theformulation. The preferred formulation contains 5mM TRIS, 75 mM NaCl, 5%sucrose, 1mM MgCl₂, 0.005% polysorbate 80 at pH 8.0 This has a pH anddivalent cation composition which is near the optimum for Ad5 stabilityand minimizes the potential for adsorption of virus to a glass surface.It does not cause tissue irritation upon intramuscular injection. It ispreferably frozen until use.

[0063] The amount of adenoviral particles in the vaccine composition tobe introduced into a vaccine recipient will depend on the strength ofthe transcriptional and translational promoters used and on theimmunogenicity of the expressed gene product. In general, animmunologically or prophylactically effective dose of 1×10⁷ to 1×10¹²particles and preferably about 1×10¹⁰ to 1×10¹¹ particles isadministered directly into muscle tissue. Subcutaneous injection,intradermal introduction, impression through the skin, and other modesof administration such as intraperitoneal, intravenous, or inhalationdelivery are also contemplated. It is also contemplated that boostervaccinations are to be provided. Following vaccination with HIVadenoviral vector, boosting with a subsequent HIV adenoviral vectorand/or plasmid may be desirable. Parenteral administration, such asintravenous, intramuscular, subcutaneous or other means ofadministration of interleukin-12 protein, concurrently with orsubsequent to parenteral introduction of the vaccine compositions ofthis invention is also advantageous.

[0064] Another aspect of this invention is the administration of theadenoviral vector containing the optimized gag gene in a prime/boostregiment in conjunction with a plasmid DNA encoding gag. To distinguishthis plasmid from the adenoviral-containing shuttle plasmids used in theconstruction of an adenovirus vector, this plasmid will be referred toas a “vaccine plasmid”. The preferred vaccine plasmids to use in thisadministration protocol are disclosed in pending U.S. patent applicationSer. No. 09/017,981, filed Feb. 3, 1998 and WO98/34640, published Aug.13, 1998, both of which are hereby incorporated by reference. Briefly,the preferred vaccine plasmid is designated V1Jns-FL-gag, whichexpresses the same codon-optimized gag gene as the adenoviral vectors ofthis invention. The vaccine plasmid backbone, designated V1Jns containsthe CMV immediate-early (IE) promoter and intron A, a bovine growthhormone-derived polyadenylation and transcriptional termination sequenceas the gene expression regulatory elements, and a minimal pUC backbone(Montgomery et al, 1993 DNA Cell Biol. 12:777-783. The pUC sequencepermits high levels of plasmid production in E. coli and has a neomycinresistance gene in place of an ampicillin resistance gene to provideselected growth in the presence of kanamycin. Those of skill in the art,however, will recognized that alternative vaccine plasmid vectors may beeasily substituted for this specific construct, and this inventionspecifically envisions the use of alternative plasmid DNA vaccinevectors.

[0065] The adenoviral vector and/or vaccine plasmids of this inventionpolynucleotide may be unassociated with any proteins, adjuvants or otheragents which impact on the recipients' immune system. In this case, itis desirable for the vector to be in a physiologically acceptablesolution, such as, but not limited to, sterile saline or sterilebuffered saline. Alternatively, the vector may be associated with anadjuvant known in the art to boost immune responses, such as a proteinor other carrier. Agents which assist in the cellular uptake of DNA,such as, but not limited to, calcium ions, may also be used toadvantage. These agents are generally referred to herein as transfectionfacilitating reagents and pharmaceutically acceptable carriers.Techniques for coating microprojectiles coated with polynucleotide areknown in the art and are also useful in connection with this invention.

[0066] The adenoviral vaccines of this invention may be administeredalone, or may be part of a prime and boost administration regimen. Amixed modality priming and booster inoculation scheme will result in anenhanced immune response, particularly is pre-existing anti-vectorimmune responses are present. This one aspect of this invention is amethod of priming a subject with the plasmid vaccine by administeringthe plasmid vaccine at least one time, allowing a predetermined lengthof time to pass, and then boosting by administering the adenoviralvaccine. Multiple primings typically, 1-4, are usually employed,although more may be used. The length of time between priming and boostmay typically vary from about four months to a year, but other timeframes may be used. In experiments with rhesus monkeys, the animals wereprimed four rimes with plasmid vaccines, then were boosted 4 monthslater with the adenoviral vaccine. Their cellular immune response wasnotably higher than that of animals which had only received adenoviralvaccine. The use of a priming regimen may be particularly preferred insituations where a person has a pre-existing anti-adenovirus immuneresponse.

[0067] This invention also includes a prime and boost regimen wherein afirst adenoviral vector is administered, then a booster dose is given.The booster dose may be repeated at selected time intervals.

[0068] A large body of human and animal data supports the importance ofcellular immune responses, especially CTL in controlling (oreliminating) HIV infection. In humans, very high levels of CTL developfollowing primary infection and correlate with the control of viremia.Several small groups of individuals have been described who arerepeatedly exposed to HIV by remain uninfected; CTL has been noted inseveral of these cohorts. In the SIV model of HIV infection, CTLsimilarly develops following primary infection, and it has beendemonstrated that addition of anti-CD8 monoclonal antibody abrogatedthis control of infection and leads to disease progression. Thisinvention uses adenoviral vaccines alone or in combination with plasmidvaccines to induce CTL.

[0069] Cellular Immunity Assays for Pre-Clinical and Clinical Research

[0070] Another aspect of this invention is an assay which measures theelicitation of HIV-1 protein, including gag-specific cellular immunity,particularly cytotoxic T-lymphocyte (CTL) responses. The “ELIspot” andcytotoxicity assays, discussed herein, measure HIV antigen-specific CD8+and CD4+ T lymphocyte responses, and can be used for a variety ofmammals, such as humans, rhesus monkeys, mice, and rats.

[0071] The ELIspot assay provides a quantitative determination ofHIV-specific T lymphocyte responses. PMBC cells are cultured in tissueculture microtiter plates. An HIV-1 gag peptide pool that encompassesthe entire 500 amino acid open reading frame of gag (50 overlapping20mer peptides) is added to the cells and one day later the number ofcells producing gamma interferon (or another selected interferon) ismeasured. Gamma interferon was selected as the cytokine visualized inthis assay (using species specific anti-gamma interferon monoclonalantibodies) because it is the most common, and one of the most abundantcytokines synthesized and secreted by activated T lymphocytes. For thisassay, the number of spot forming cells (SPC) per million PBMCs isdetermined for samples in the presence and absence (media control) ofpeptide antigens. This assay may be set up to determine overall Tlymphocyte responses (both CD8+ and CD4+) or for specific cellpopulations by prior depletion of either CD8+ or CD4+ T cells. Inaddition, ELIspot assays, or variations of it, can be used to determinewhich peptide epitopes are recognized by particular individuals.

[0072] A distinguishing effector function of T lymphocytes is theability of subsets of this cell population to directly lyse cellsexhibiting appropriate MHC-associated antigenic peptides. This cytotoxicactivity is most often associated with CD8+ T lymphocytes but may alsobe exhibited by CD4+ T lymphocytes. We have optimized bulk culture CTLassays in which PBMC samples are infected with recombinant vacciniaviruses expressing antigens (e.g., gag) in vitro for approximately 14days to provide antigen restimulation and expansion of memory T cellsthat are then tested for cytoxicity against autologous B cell linestreated either with peptide antigen pools. Specific cytotoxicity ismeasured compared to irrelevant antigen or excipient-treated B celllines. The phenotype of responding T lymphocytes is determined byappropriate depletion of either CD8+ or CD4+ populations prior to thecytotoxicity assay. This assay is semi-quantitative and is the preferredmeans for determining whether CTL responses were elicited by thevaccine.

[0073] The following non-limiting Examples are presented to betterillustrate the invention.

EXAMPLES EXAMPLE 1 Construction of replication-defective FG-Adexpressing HIV gag antigen

[0074] Starting vectors

[0075] Shuttle vector pHCMVIBGHpA1 contains Ad5 sequences from bp1 to bp341 and bp 3534 to bp 5798 with a expression cassette containing humancytomegalovirus (HCMV) promoter plus intron A and bovine growth hormonepolyadenylation signal.

[0076] The adenoviral backbone vector pAdE1-E3- (also named as pHVad1)contains all Ad5 sequences except those nucleotides encompassing the E1and E3 region.

[0077] Plasmid pV1JNStpaHIVgag contains tPA secretory signal sequencefused to the codon-optimized HIV gag nucleotides under the control ofHCMV promoter plus intron A. It is described in pending U.S. patentapplication Ser. No. 09/017,981, filed Feb. 3, 1998 and WO98/34640,published Aug. 13, 1998, both of which are hereby incorporated byreference.

[0078] Plasmid pV1R-FLHIV gag (also named as pV1R-HIVgag-opt) containscodon-optimized full-length HIV gag under the control of the HCMVpromoter plus intron A.

[0079] Construction of Ad5tpaHIVgag

[0080] 1. Construction of adenoviral shuttle plasmid pA1-CMVI-tpaHIVgagcontaining tPAgag under the control of human CMV promoter and intron A.

[0081] The tPAgag insert was excised from pV1JNS-tPAgag by restrictionenzymes PstI and XmaI, blunt-ended, and then cloned into EcoRV digestedshuttle vector pHCMVIBGHpA1. The orientation of the transgene and theconstruct were verified by PCR using the insert specific primershCMV5′-4 (5′TAG CGG CGG AGC TTC TAC ATC 3′ SEQ.ID.NO.2) and Gag3′-1(5′ACT GGG AGG AGG GGT CGT TGC 3′SEQ.ID.NO.3), restriction enzymeanalysis (RcaI, SspBI), and DNA sequencing spanning from CMV promoter tothe initiation of the gag.

[0082] 2. Homologous recombination to generate shuttle plasmid form ofrecombinant adenoviral vector pAd-CMVI-tpaHIVgag containing tpaHIVgagexpression cassette.

[0083] Shuttle plasmid pA1-CMVI-tpaHIVgag was digested with restrictionenzymes BstZ17 and SgrA1 and then co-transformed into E. coli strainBJ5183 with linearized (ClaI digested) adenoviral backbone plasmidpAdE1-E3-. One colony was verified by PCR analysis. The vector wastransformed to competent E. coli HB101 for large quantity production ofthe plasmid.

[0084] 3. Generation of recombinant adenovirus Ad.CMVI-tpaHIVgag in 293cells.

[0085] The shuttle plasmid was linearized by restriction enzyme PacI andtransfected to 293 cells using CaPO₄ method (InVitrogen kit). Ten dayslater, 10 plaques were picked and grown in 293 cells in 35-mm plates.PCR analysis of the adenoviral DNA indicated 10 out of 10 virus werepositive for gag.

[0086] 4. Evaluation of large scale recombinant adenovirusAd.CMVI-tpaHIVgag

[0087] Clone No.9 was grown into large quantities through multiplerounds of amplification in 293 cells. One lot yielded of 1.7×10¹²particles and a second lot yielded 6.7×10¹³ particles. The viral DNA wasextracted by proteinase K digestion and confirmed by PCR and restrictionenzyme (HindIII) analysis. The expression of tpaHIVgag was also verifiedby ELISA and Western blot analysis of the 293 or COS cells infected withthe recombinant adenovirus. The recombinant adenovirus was used forevaluation in mice and rhesus monkeys.

[0088] Construction of Ad5.FHIVgag

[0089] 1. Construction of adenoviral shuttle plasmid pA1-CMVI-FLHIVgagcontaining full length HIVgag under the control of human CMV promoterand intron A.

[0090] The FLHIVgag insert was excised from pV1R-FLHIVgag by restrictionenzyme BglII and then cloned into BglII digested shuttle vectorpHCMVIBGHpA1. The orientation and the construct were verified by PCRusing the insert specific primers (hCMV5′-4 and Gag3′-1), restrictionenzyme analysis, and DNA sequencing.

[0091] 2. Homologous recombination to generate plasmid form ofrecombinant adenoviral vector pAd-CMVI-FLHIVgag containing FLHIVgagexpression cassette.

[0092] Shuttle plasmid pA1-CMVI-FLHIVgag was digested with restrictionenzymes BstZ17 and SgrA1 and then co-transformed into E. coli strainBJ5183 with linearized (ClaI digested) adenoviral backbone plasmidpAdE1-E3-. Colonies #6 and #7 were verified by PCR analysis. The vectorswere transformed to competent E. coli HB101 for large quantityproduction of the plasmid. The plasmids were verified by HindIIIdigestion.

[0093] 3. Generation of recombinant adenovirus Ad.CMVI-FLHIVgag in 293cells.

[0094] The pAd plasmids were linearized by restriction enzyme PacI andtransfected to 293 cells using Lipofectamine (BRL). Two weeks later, 6viruses (#6-1.1, 6-1.2, 6-1.3, 7-1.1, 7-1.2, 7-1.3) were picked andgrown in 293 cells in 35-mm plates. PCR analysis using the insertspecific primers (hCMV5′-4 and Gag3′-1) of the adenoviral DNA verifiedthe presence of HIV gag. p0 4. Evaluation of large scale recombinantadenovirus Ad.CMVI-FHIvgag

[0095] Virus clone #6-1 was grown into large quantities through multiplerounds of amplification in 293 cells. The viral DNA was extracted byproteinase K digestion and confirmed by PCR, restriction enzyme(HindIII, Bgl II, Bst E II, Xho I) analysis. A partial sequencingconfirmed the junction between CMV promoter and the 5′end of HIV gaggene. The expression of FLHIVgag was also verified by ELISA and Westernblot analysis of the 293 or COS cells infected with the recombinantadenovirus. The recombinant adenovirus was used for evaluation in miceand rhesus monkeys.

[0096] Construction of FG adenovirus FL gag. The full-length (FL)humanized gag gene was ligated into an adenovirus-5 shuttle vector,pHCMVIBGHpA1, containing Ad5 sequences from bp 1 to bp 341 and bp 3534to bp 5798 with a expression cassette containing human CMV promoter plusintron A and bovine growth hormone polyadenylation signal. Theorientation was confirmed by restriction enzyme digestion analysis andDNA sequencing. Homologous recombination in E. coli was employed usingthe shuttle plasmid, pA1-CMVI-FLHIVgag, and adenoviral backbone plasmid,pAdE1-E3-, to generate a plasmid form of the recombinant adenoviruscontaining the expression regulatory elements and FL gag gene,pAd.CMVI-FHIVgag. Appropriate plasmid recombinants were confirmed byrestriction enzyme digestion.

[0097] The pAd plasmid containing the gag expression cassette waslinearized by restriction enzyme PacI and transfected to 293 cells (orPER.C6 cells for clinical development candidates) using Lipofectamine(BRL). Two weeks later, 6 viruses were picked and grown in 293 cells in35-mm plates. PCR analysis using the insert specific primers (hCMV5′-4and Gag3′-1) of the adenoviral DNA verified the presence of HIV gag.Virus clone #6-1 was grown into large quantities through multiple roundsof amplification in 293 cells. The viral DNA was extracted by proteinaseK digestion and confirmed by PCR, restriction enzyme (HindIII, BglII,BstEII, XhoI) analysis. A partial sequencing confirmed the junctionbetween CMV promoter and the 5′end of HIV gag gene. Restriction enzymeanalysis demonstrated that the viral genome was stable over the courseof these passages.

[0098] The expression of HIV gag was verified by ELISA and Western blotanalysis of the 293 or COS cells infected with the recombinantadenovirus.

EXAMPLE 2 Immunogenicity/Preclinical Efficacy

[0099] The “ELIspot” Assay

[0100] The ELIspot assay is a quantitative determination of IV-specificT lymphocyte responses by visualization of gamma interferon secretingcells in tissue culture microtiter plates one day following addition ofan HIV-1 gag peptide pool that encompasses the entire 500 amino acidopen reading frame of gag (50 overlapping 20mer peptides) to PBMCsamples. The number of spot forming cells (SPC) per million of PBMVs isdetermined for samples in the presence and absence (media control) ofpeptide antigens. The assay may be set up to determine overall Tlymphocyte responses (both CD8+ and CD4+) or for specific cellpopulations by prior depletion of either CD8+ or CD4+ cells. Inaddition, the assay can be varied so as to determine which peptideepitopes are recognized by particular individuals.

[0101] Cytotoxic T Lymphocyte Assays

[0102] In this assay, PBMC samples are infected with recombinantvaccinia viruses expressing gag antigen in vitro for approximately 14days to provide antigen restimulation and expansion of memory T cells.The cells are then tested for cytotoxicity against autologous B celllines treated with peptide antigen pools. The phenotype of responding Tlymphocytes is determined by appropriate depletion of either CD8+ orCD4+ cells.

[0103] A. Immune Responses to FG Adenovirus 5 FLgag Vaccine in Rodents

[0104] Adenovirus vectors coding for the gag antigen have consistentlyproduced significantly stronger cellular immune responses than plasmidvectors in rodent species. Table 1 (below) shows ELIspot data from micevaccinated with Ad5FLgag in comparison with plasmid DNA. Spleens fromfive mice were pooled and the number of gag peptide-specificinterferon-gamma secreting cells was determined. TABLE 1 Comparison ofplasmid and adenovirus vaccination in mice SFC/10⁶ splenocytes Post 1stPost 2nd Vaccination Vaccination 10 μg plasmid 68 324 10⁵ Ad5Flgag 18170 10⁸ Ad5Flgag 530 5600

[0105] Similar enhancements in the cellular responses to gag were alsoseen in Fischer rats. FIG. 1 shows the ELIspot data from individual ratsvaccinated with 10¹⁰ particles of adenovirus Ad5FLgag or with 0.4 mg FLgag plasmid. The mean response after one vaccination was 10-fold higherwith adenovirus compared to plasmid. Both vaccines gave a boosted signalafter a second vaccination, with the adenovirus vaccine signal 5-foldhigher than the plasmid signal.

[0106] B. Immune Responses to FG Adenovirus 5 FL gag Vaccine in RhesusMonkeys

[0107] Comparative in vivo expression of DNA vs. FGAd5 encoding areporter gene. Adenovirus and plasmid vectors expressing the secretedalkaline phosphatase (SEAP) as a reporter gene were injected into rhesusmonkeys to compare the levels of antigen produced by the two forms ofvaccination as shown below. FIG. 2 shows that at the highest possibleplasmid dose (5 mg), the antigen levels are 1,000-fold lower than thelevels achieved using 10¹⁰ particles of adenovirus, a dose which is tenfold lower than the maximum proposed clinical dose.

[0108] FG adenovirus-5 FLgag vaccinations of rhesus monkeys. ThreeRhesus monkeys were vaccinated at 0, 8, and 24 weeks with 10¹¹ particlesof FG adenovirus-5tPAgag, an adenoviral vector containing a form of thegag gene with a leader peptide from the tissue-specific plasminogenactivator gene at the amino terminus. Data were collected starting at 20weeks.

[0109]FIG. 3 shows that all three monkeys had developed strong bulkculture cytotoxicity responses against gag peptide sensitized autologousB cell lines following in vitro restimulation using vaccinia-gag for twoweeks. These responses were persistent at all time points testedalthough it is unclear whether the final inoculation at 24 weeksimproved the cytotoxicities. In all cases killing is observed with atleast one partial peptide pool (i.e. 25 peptides from amino terminus, 25peptides from the carboxyterminus of gag). In every case killing isobserved with the full peptide pool (all 50 peptides spanning thefull-length gag). ELIspot assays showed high levels of gamma interferonsecreting cells (approximately 200-1000 SFC/million PBMCs) over thecourse of these experiments, and CD4/8 depletion studies indicated thatmost responding cells were CD8 T lymphocytes, although most vaccinesalso had significant CD4 T cell responses.

[0110] Immunization of rhesus monkeys with FG adenovirus-5 FLgag. Usinga protocol similar to that described above, monkeys were vaccinated witha FG adenovirus-5 construct encoding a full-length gag gene (without thetPA leader peptide). This experiment compared a dose titration ofvaccine as well as needle vs. biojector (a needleless injector) deliveryat most doses. A third feature of this experiment addresses the concernsraised above about the possible negative effects that prior adenovirusimmunity may have on adenoviral vector mediated vaccine responses.

[0111] While approximately 150 rhesus sera have been tested foranti-adenovirus-5 neutralizing antibody responses, no significant titershave been detected. Rhesus are a poor host for this viral strain, whileabout 40-60% of humans have significant neutralizing antibody responses(titers from 10-500). For this experiment, two groups of monkeys (6 and7) were pre-exposed to FG adenovirus-SEAP vectors thrice and once,respectively, generating a range of neutralizing antibody responses inthese monkeys that encompass the range observed in humans.

[0112] FIGS. 4A-I show bulk culture cytotoxicity responses of thesevaccines at 8 weeks post a single immunization. All monkeys (groups 1-5,FIGS. 4A-E) that had not been previously exposed to adenovirus-5 showedsignificant gag-specific cytotoxic responses at all doses using eitherbiojector or needle while ⅚ vaccines that had been preexposed toadenovirus showed cytotoxic responses (groups 6-7, FIGS. 4F-G). Controlanimals have remained consistently negative in these assays (e.g., group8, FIG. 4H).

[0113] Anti-gag ELIspot responses were also measured in all monkeys ateight weeks. Table 2, below is a summary of these responses that showthat nearly all vaccines developed significant gamma-interferonresponses to this vaccine, although prior exposure to adenovirus reducedresponse levels, and a dose response appears to have been obtained withthe highest doses giving the best responses. In addition, in thisexperiment (as well as an independent experiment) no difference wasobserved for needle vs. biojector delivery of vaccine. CD4 T celldepletion of these samples showed that the ELIspot responses are largelydue to CD8 T cells. TABLE 2 Anti-gag ELIspot Responses of Rhesus MonkeysImmunized with FG adenovirus FLgag vaccine. Rhesus Prior AdenoSFC/million PBMCs Group # Injection Exposure media gag pool H 1 96R044Biojector none 6 663 ″ 96R045 ″ ″ 0 665 ″ 96R046 ″ ″ 5 893 2 96R047Biojector none 1 20 ″ 96R048 ″ ″ 1 104 ″ 96R049 ″ ″ 0 38 3 96R050Biojector none 4 18 ″ 96R051 ″ ″ 1 14 ″ 96R052 ″ ″ 10 48 4 96R053 Needlenone 1 410 ″ 96R054 ″ ″ 0 125 ″ 96R057 ″ ″ 3 186 5 96R058 Needle none 193 ″ 96R060 ″ ″ 1 41 ″ 96R062 ″ ″ 0 6 6 940125 Biojector 3 × 10¹⁰ FG 1565 aden-5 ″ 940132 ″ ″ 11 39 ″ 940149 ″ ″ 29 93 7 940145 Biojector 1 ×10¹⁰ FG 4 258 adeno-5 ″ 940147 ″ ″ 15 578 ″ 940217 ″ ″ 23 55 8 96R063none none 0 0 ″ 96R004 ″ ″ 0 1

[0114] These and other data show that higher doses of FG adenovirusvaccines elicited ELIspot responses as high as 800-1000 SFC/millionPBMCs (see Table 2). These responses are approximate 5-10 fold higherthan those obtained using the highest doses of DNA gag vaccines afterfour injections over a six month time frame (see Table 3 below)indicating that FG adenovirus vaccines are much more potent than DNAvaccines. Importantly, these data also support the finding that repeatedinjection of adenoviral vector remain effective although somewhatattenuated in the presence of host immune response to adenovirus.

[0115] Combined DNA and FG adenovirus vaccinations in rhesus.

[0116] DNA priming may enhance the cellular immune response to gaginduced by adenovirus vaccination as shown below. Three rhesus monkeyswhich had been vaccinated four times with 1 mg of gag plasmid wereboosted 4 months following the final DNA shot with 10¹¹ particles of FGAd ELgag. The cellular immune responses (measured by ELIspot and denotedas SFC/million PBMCs) to gag peptides in the monkeys primed with DNA andboosted with adenovirus appear significantly higher than adenovirusvaccination alone. The use of a DNA priming regimen may be particularlyadvantageous in humans who have preexisting anti-adenovirus immuneresponses.

[0117] Table 3. ELIspot Responses in Rhesus Monkeys After Combined DNAand FG adenovirus gag Vaccinations. Week 20 Week 28 Monkey (2 (3 #Priming Vaccine injections) injections) DNA prime/ 92 × 004 DNAAd5tPAgag 106 781 Ad boost 93 × 027 DNA Ad5tPAgag 88 660 93 × 023 DNAAd5tPAgag 560 609 DNA prime/ 93 × 008 DNA DNA 344 285 DNA boost 93 × 012DNA DNA NA NA 93 × 016 DNA DNA 106 99 Naive/Ad 92 × 024 None Ad5tPAgag373 898 92 × 012 None Ad5tPAgag 276 413 94 × 025 None Ad5tPAgag 531 1275Control 088R None None 5 84 115Q None None 0 8

[0118] TABLE 4 Anti-gag antibody titer (mMU/ml) in Rhesus Monkeys AfterDNA and FG adenovirus gag Vaccinations. Week 8 Week 20 Week 28 MonkeyWeek (1 (2 (3 Week # 0 injection) injections) injections) 40 DNA prime/92 × 004 25 7616 10133 12170 15892 Ad boost 93 × 027 114 36666 2052395114 31437 93 × 023 41 11804 12485 38579 17422 DNA prime/ 93 × 008 1581689 817 3882 1626 DNA boost 93 × 012 <10 512 216 722 132 93 × 016 20305 451 2731 735 Naive/Ad 92 × 024 <10 2454 11460 15711 7449 92 × 012<10 2161 5154 27029 8856 94 × 025 14 5852 19159 45990 37586

[0119] C. Determination of HIV-Specific T Lymphocyte Responses in HIV+Humans

[0120] In order to qualify the CTL assays, PBMCs from HIV-1-infectedpatients, classified as long-term nonprogessors (LTNPs) due to theirability to maintain low levels of systemic viremia and high CD4⁺ T cellcounts over a period of years, were used to measure systemic specificCTL responses. As discussed above, several studies have reported thatthe presence of HIV-1-specific CTL responses in infected individualsappears to correlate well with maintenance of disease-free infection.

[0121] Over the course of numerous independent experiments using PBMCsobtained from approximately 40 LTNPs at five different clinical centers,these HIV-infected individuals generally exhibited strong gag-specificELIspot and cytotoxicity responses that are predominantly mediated byCD8+ T lymphocytes (CD4+ responses are typically extremely low orundetectable in HIV+ individuals). The overall gag-specific ELIspotresponses determined in these experiments are summarized below:

[0122] HIV+ ELIspot Response Summary

[0123] mean (±SD) SFC/million PBMCs (+gag peptides)=980±1584

[0124] mean (±SD) SFC/million PBMCs (media control)=24±21

[0125] Similar experiments using PBMCs from 16 HIV seronegativeindividuals did not show significant gag-specific ELIspot orcytotoxicity responses. These ELIspot responses are summarized below:

[0126] HIV Seronegative ELIspot Response Summary

[0127] mean (±SD) SFC/million PBMCs (+gag peptides)=19±28

[0128] mean (±SD) SFC/million PBMCs (media control)=10±14

[0129] The ELIspot assay provides a quantitative determination ofHIV-specific T lymphocyte responses by visualization of gammainterferon-secreting cells in tissue culture microtiter plates one dayfollowing addition of an HIV-1 gag peptide pool that encompasses theentire 500 amino acid open reading frame of gag (50 overlapping 20merpeptides) to PBMC samples. Gamma interferon was selected as the cytokinevisualized in this assay (using species specific anti-gamma interferonmonoclonal antibodies) because it is the most common, and one of themost abundant cytokines synthesized and secreted by activated Tlymphocytes. For this assay, the number of spot forming cells (SPC) permillion PBMCs is determined for samples in the presence and absence(media control) of peptide antigens. This assay may be set up todetermine overall T lymphocyte responses (both CD8+ and CD4+) or forspecific cell populations by prior depletion of either CD8+ or CD4+ Tcells. In addition, ELIspot assays, or variations of it, can be used todetermine which peptide epitopes are recognized by particularindividuals.

[0130] A distinguishing effector function of T lymphocytes is theability of subsets of this cell population to directly lyse cellsexhibiting appropriate MHC-associated antigenic peptides. This cytotoxicactivity is most often associated with CD8+ T lymphocytes but may alsobe exhibited by CD4+ T lymphocytes. We have optimized bulk culture CTLassays in which PBMC samples are infected with recombinant vacciniaviruses expressing antigens (e.g., gag) in vitro for approximately 14days to provide antigen restimulation and expansion of memory T cellsthat are then tested for cytoxicity against autologous B cell linestreated either with peptide antigen pools. Specific cytotoxicity ismeasured compared to irrelevant antigen or excipient-treated B celllines. The phenotype of responding T lymphocytes is determined byappropriate depletion of either CD8+ or CD4+ populations prior to thecytotoxicity assay. This assay is the best means for determining whetherCTL responses were elicited by the vaccine.

EXAMPLE 3 Clinical Trials

[0131] The safety and efficacy of a first generation adenovirus type 5carrying an optimized gag gene alone and as part of a prime/boostprotocol with a gag DNA plasmid are tested.

[0132] In the initial trial, subjects receive either 1 mg or 5 mg HIVgag DNA on a 0, 1, 2 month schedule. Equal number so of Ad5 seropositiveand seronegative subjects are involved in the study.

[0133] In a second trial, Ad5 seropositive and seronegative individualsreceive either 10⁷ or 10⁹ particles per dose on a 0, 6 month schedule.Some of the individuals who have received a single dose of 10⁹ particledof Ad5gag will also receive three injections of HIV gag DNA with 10¹¹particles of Ad5 gag. Also, individuals who are Ad5 seropositive andseronegative naive individuals will receive 10¹¹ particles on a 0, 6month schedule.

[0134] Safety and immunogenicity parameters: Each individual is bled forserum prior to day 0 to determine Ad5 neutralization titers and forPBMCs to establish B-LCL lines for bulk CTL determinations. On day 0 and4 weeks following each dose of Ad5 gag, PBMCs will be drawn to determineCTL using bulk CTL and ELIspot assays. Immunogenicity will also bemeasured at later time points to assess persistence of response.Clinical Study Design Summary 1. Phase I study of HIV gag DNA Priming(plasmid only) Vaccine Placebo DNA DNA Ad5 Sero- GROUP (n) (n) doseregimen status 1 18 3 1 mg 0, 1, 2 + 2 18 3 1 mg 0, 1, 2 − 3 18 3 5 mg0, 1, 2 + 4 18 3 5 mg 0, 1, 2 = TOTAL 72 12 2. Dose ranging study of AD5gag Vaccine Placebo Ad5 Ad5 sero Group (n) (n) Dose status 1 8 2 10⁷  +2 8 2 10⁷  − 3 8 2 10⁹  + 4 8 2 10⁹  − 5 15 2 10¹¹ + 6 15 2 10¹¹ − TOTAL62 12 3. Boosting of gag DNA by Ad5 gag Ad5 Ad5 sero- DNA DNA GroupVaccine Placebo dose status dose regimen 1* 15 2 10¹¹ + 1 mg 0, 1, 2 2*15 2 10¹¹ − 1 mg 0, 1, 2 3* 60 2 10¹¹ + 5 mg 0, 1, 2 4* 60 2 10¹¹ − 5 mg0, 1, 2

EXAMPLE 4 Longevity of CMI Responses in Monkeys Vaccinated withAd5-Flgag

[0135] 69 weeks post the last vaccination (two injections at week 0 and24), PBMC isolated from these monkeys were tested in unfractionated orCD4 T cell-depleted ELISPOT assays. The results in FIG. 9 show that highlevel of CMI responses were maintained in these vaccinees, and theresponses were predominantly CD8⁺ as those detected following the 2^(nd)injection.

EXAMPLE 5 Prime/Boost Regimens Utilizing V1JnsFLgag and/or Ad5FLgag inRhesus Monkeys

[0136] Rhesus monkeys immunized with HIV gag DNA vaccine formulated withAlPO4 or the POP-POE copolymer CRL1005 (CytRx; Atlanta, Ga.) at 7.5 mg(injected at weeks 0, 4 and 8) were boosted with low dose adenovirus(10{circumflex over ( )}7 particles) at week 24. The strategy of usingthe low dose adenovirus was adopted to mimic the prevalence ofanti-adeno neutralizing Abs in vaccinees, based on our early observationthat the presence of anti-adeno Ab titers in rhesus monkeys willeffectively reduce the vaccination dose by two to three orders ofmagnitudes. It has also been shown that this dose of Ad5-FLgag is notefficient in priming CMI responses in monkeys, judged by the respectiveELISPOT responses. The prime-boost results showed, however, that thislow dose of Ad5 vaccines is highly efficient in boosting immuneresponses primed with DNA vaccines. The prime-boost strategy outlinedhere provides an alternative vaccination regimen to overcome theanti-Ad5 seroprevalence in human population. FIG. 10 shows CMI responsesprior to and subsequent to the week 24 boost with Ad5-Flgag. These datashow that adjuvant formulations of DNA primed CMI responses moreefficiently than naked DNA in saline.

[0137]FIG. 11 shows long term CMI responses for an HIV gag DNA vaccine(0, 4 and 8 weeks) and Ad5FLgag (single prime at T=0) which were boostedwith 10⁷ particles of Ad5FLgag.

[0138]FIG. 12 shows data that compares the cellular immune response forvarious prime/boost regimens. Three groups shown in the left threepanels received adenoviral vector prime and adenoviral vector boost at10{circumflex over ( )}11 particle dose (week 0 and 24). The fiveDNA-primed (week 0, 4, 8) groups shown in the right five panels receivedadenoviral vector at either 10{circumflex over ( )}7 or 10{circumflexover ( )}11 particles (week 24). Different CRL-1005 formulations weretested for their ability to enhance the immunogenicity of DNAvaccination (D113, D117 and D118: D113 is 5 mg/ml DNA, 7.5 mg CRL1005 inPBS; D117 is 5 mg/ml DNA, 22.5 mg CRL1005 in PBS; and D118 is 7.5 mg/mLCRL1005, 0.5 mM BAK and 5 mg/mL DNA in PBS. DNA/POP-POE/BAK formulationsare disclosed in U.S. Provisional Application Serial Nos. 60/214,824 and60/213622, filed Jun. 28, 2000 and Jun. 23, 2000, respectively; both ofwhich are hereby incorporated by reference). The data showed: 1)pre-existing anti-adeno titers in vaccinees reduce the priming andboosting efficacy of the Adenoviral vector expression HIV1 gag; 2) DNApriming can overcome the low dose adenoviral vector boost; 3) efficiencyof priming with DNA vaccine can be improved with adjuvanting ofCRL-1005; and 4) CRL-1005 DNA priming coupled with low dose adenoviralvector boosting can induce CMI responses equivalent to those seen inadenovirus-naïve monkeys received 10{circumflex over ( )}11 particles ofthe adenoviral vector expressing HIV-1 gag. Therefore, while there maybe attenuation of the response to a defective adenovirus gag vectormediated by pre-existing neutralizing antibodies, a portion of thepresent invention, in view of this data, relates to the ability toovercome any such attenuation by a prime/boost immunization regime witha priming by a selected DNA plasmid formulation expressing the HIV-1 Gagantigen, followed by a boosting with an adenovirus vector expressing theGag antigen.

EXAMPLE 6 Evaluation of SIVmac239 Gag-Based Vaccines for Protection ofRhesus Macaques Against Challenge of SHIV89.6P

[0139] Materials and Methods—Animals—Fifteen rhesus monkeys (Macacamulatta) were assigned in five groups received vaccines, while 6 wereassigned as naïve control monkeys that did not receive any vaccination.All monkeys were typed for Mamu-A*01 allele expression according toPCR-SSP methodology. Briefly, genomic DNA was extracted from monkey PBMCor B lymphoid cells using QiaAmp DNA Kit (Qiagen), and about 20 ug to100 ng of DNA was used for PCR reactions. Two pairs of primers wereused, one for detection of a fragment within Mamu-A*01 allele, and onefor a fragment within Mamu-DRB region to serve as positive control forPCR reaction. All animals that tested positive in PCR reaction wereconfirmed by sequencing methods.

[0140] Vaccines and vaccination regimen—Codon optimized SIVmac239 p55gag gene (no pol sequences) was constructed with annealing of a seriesof overlapping oligonucleotides. The authenticity of the synthetic genewas confirmed with DNA sequencing. The gene was cloned into V1Jns, asdisclosed herein. The same synthetic gene was cloned into either MVA oradenovirus vectors. Regarding recombinant MVA vector construction, thecodon optimized SIV gag gene with an authentic Kozak sequence (GCCACC)in front of ATG was PCR amplified and cloned into the pSC59 shuttlevector. The vector was designed to insert the transgene fragment intothe viral thymidine kinase region, and to drive the transgene from asynthetic early/late promoter. The recombinant vector was selectedthrough plaque assay six times, and confirmed each time based onimmunostaining method. The selected clones were expanded, purified, andtransgene expression was confirmed by Western blot analysis. Theadenovirus vector was generated by cloning the codon optimized SIV gaggene fragment downstream of the CMVpromoter with noIntron A region intoan adenoviral shuttle vector. All constructs were confirmed forexpression by Western blot analysis. The plasmid DNA vaccine expressedthe codon optimized SIV gag gene utilizing a CMV promoter in a V1Jnsplasmid vector backbone. Therefore, the vaccine groups were as follows:

[0141] (1) Naïve Controls (MamuA1+and MamuA1—monkeys);

[0142] (2) MVA SIV gag (10⁹ pfu);

[0143] (3) Ad5 SIV gag (10¹¹ viral particles ˜10⁹ pfu);

[0144] (4) SIV gag DNA (5 mg);

[0145] (5) SIV gag DNA (5 mg)+CRL-1005 (CytRx, Atlanta, Ga.)

[0146] (6) SIV gag DNA (5 mg)+Alum/MPL

[0147] Plasmid DNAs were injected at weeks 0, 4, 8, 32. MVA andAdenovirus vectors were injected at weeks 0, 6, 32. The monkey were SHIVchallenged (50 MID₅₀ intravenously) at week 44, which was 12 weeks postlast immunization. SHIV 89.6P is a HIV-1/SIV-1 chimera (HIV-1 tat, rev,env; with a SIV backbone). HIV env of SHIV 89.6P is derived from aprimary, dual tropic viral isolate, which is rendered highly viremic andpathogenic by serial in vivo passage. SHIV 89.6 causes acute CD4lymphopenia (typically in 7-21 days, while causing AIDS and death in 1-2years.

[0148] Tetramer staining—The method for preparing p11C tetramer reagentand staining whole blood is as follows: PE-labeled tetramericMamu-A*01/peptide complexes was used in conjunction with PerCP-labeledanti-human CD8⁺ (SK1; Becton Dickinson), and APC-labeled anti-rhesusmonkey CD3 (FN18) monoclonal antibodies to stain peptide-specific CD8⁺ Tcells. A sample of 100 μl whole blood from the vaccinated monkeys wasdirectly stained with these reagents, lysed, washed, and fixed. Sampleswere analyzed through flow cytometry on FACScalibur [Becton Dickinson(BD), San Jose, Calif.], and gated CD3⁺CD8⁺ T cells were examined forstaining with tetrameric Mamu-A*01/p11C, Mamu-A*01/p41A, orMamu-A*01/p68A complexes. For each sample, 30,000 gated CD3+/CD8+lymphocyte events were collected and analyzed on CellQuest program (BD).

[0149] ELSIPOT assay—Sterile 96-well microtiter plates with well bottomsof polyvinylidene difluoride (PVDF, MAIP S45, Millipore) were coatedovernight at 4° C. with 100 μl/well of mouse anti-human IFN-γ monoclonalantibody (R&D Systems). The antibody was diluted to 10 μg/ml in sterilePBS. Coated plates were washed four times with sterile PBS and blocked 2hours at 37° C. with 200 μl/well of R10 complete medium (RPMI-1640 plus10% heat-inactivated fetal bovine serum plus supplements). Blockingbuffer was decanted and 100 μl/well of rhesus PBMC diluted in R10 wereadded to result in 2×10⁵ and 1×10⁵ cells/well. Synthetic peptideantigens were added to the PBMC wells in duplicate at a finalconcentration of 2.5 μg/ml per peptide. Peptide-free DMSO diluentmatching the DMSO concentration in the peptide solutions was used as anegative control (medium control). The peptide pools were composed of20-mer peptides overlapping by 10 amino acids, representing N-terminalor C-terminal half of SIV gag (SIVmac239) and env (89.6P, KB9 clone)gp120 regions. The peptides containing p11C epitope was deleted from theSIV gag peptide pool for the purpose of detecting other epitopes besidesp11C. Plates were incubated 24 hours in a humidified CO₂ incubator at37° C. and then washed 7 times with PBS containing 0.05% Tween 20(Sigma). Biotinylated goat anti-human IFN-γ monoclonal antibody (R&DSystems catalog #BAF285) was diluted to 0.1 μg/ml in assay diluentconsisting of PBS and 5% heat inactivated fetal bovine serum (FBS,HyClone) and 0.005% Tween 20. Diluted antibody was added to the platesat 100 μl/well and incubated overnight at 4° C. After washed 7 timeswith PBS/Tween, 100 μl/well of alkaline phosphatase-conjugatedstreptavidin (B-D PharMingen, San Diego, Calif.) at 1:2500 in assaydiluent was added to each well. Plates were incubated 2 hours at roomtemperature and washed 7 times with PBS/Tween. To develop the spots, 100μl/well of precipitating alkaline phosphatase substrate NBT/BCIP(Pierce) was added to each well and incubated at room temperature untilspots became visible, usually 5-10 minutes. Substrate was decanted andplates rinsed three times with deionized water. After plates hadair-dried overnight, spots were counted with a stereomicroscope using amagnification of 20-25X. The number of spots per well at each cell inputwas normalized per 1×10⁶ cells and averaged for each sample and antigen.

[0150] Cytotoxicity assay—the methods describing generation of Blymphoblastoid cell lines and bulk culture cytotoxicity assay havedescribed previous (JV. Accepted). Instead of using vaccinia virus forrestimulation, recombinant Ad5-SIVgag was used for CTL cultures fromMVA-SIVgag-vaccinated animals.

[0151] Results—Improved expression is shown by utilizing a SIV gag genecodon optimized for expression in mammalian systems. The SIVmac 239 gagopen reading frame was optimized in this fashion for expression. Therewere about 60% nucleotide changes in the synthetic gene, correspondingto about 20% of codons. The improvement in expression of the optimizedgene in comparison of wild type SIVmac 239 gag was confirmed in Westernblot analysis.

[0152] The other vaccine modalities used in this study include two viralvectors, adenovirus vector which has both E1 and E3 regions deleted, andan MVA vector which has the transgene inserted at the TK region. Thetransgene in MVA vector is driven by a synthetic early/late promoter.The transgene in the adenoviral vector is driven by CMVie promoter andenhancer without intron A region.

[0153] Twenty one monkeys were assigned in this study, allocated intoone naïve control group of six monkeys, and five vaccination groups with3 monkeys per group. All monkeys were typed for Mamu-A*01 allele, andall monkeys in the vaccination groups except one monkey in the DNA groupwere Mamu-A*01 monkeys. The expression of this MHC allele facilitatedthe analysis of CD8 T cell immune response to vaccination with atetramer reagent directed against a prevalent SIV gag epitope, p11C. Toinsure the appropriate priming, all viral vaccine vectors wereadministrated twice at week 0 and 6, and all DNA vaccines three times atweek 0, 4, and 8. After a long rest of at least three months, allmonkeys were boosted with the same dose of priming modality.

[0154] Pre-challenge immunogenicity study—FIGS. 13A-E shows thelongitudinal p11IC-specific tetramer staining results for all Mamu-A*01monkeys up to one week before challenge. All results are expressed as %CD3⁺CD8⁺ positive T cell population. Following two priming injections,all three monkeys in the MVA group showed significant tet positivestaining with the highest one reaching 1.6%, and the average close to0.7%. The response in this group gradually declined, and was close todetection limit right before the boost (0.1%). However, the boostinjection with the same dose MVA did not improve the overall percentageof tet positive staining, and the peak of responses at two weeks postboost injection in all three monkeys is lower than their initial heightof staining following priming injections. Before challenge at week 44,all three monkeys are stained positive just above 0.1%. The tetramerstaining in the adenovirus-immunized group showed much higher staining,with the average of the staining at over 2% following two priminginjections. The responses reached stead level of about 0.3 to 1.2%before the boost, and were significantly boosted with staining at 3 to5.5% in all three monkeys. The staining was stead at 1-2% beforechallenge. All DNA vaccine groups received three priming injections, andall monkeys demonstrated detectable tetramer staining except theMamu-A*01 negative monkey T282. The most noticeable group is the CRL1005adjuvanted group, with a monkey reaching 2% tet staining followingpriming injections (S202). The results from MPL/alum group are similarto those of the DNA/saline group. Following boost injection, allvaccinees are significantly boosted, with average staining in each group2-3-fold higher than their respective peak staining following priminginjections. At week 37 (one week before challenge), the DNA/CRL1005group maintained about 0.3 to 0.7% tetramer positive staining, whereasboth DNA in saline and MPL/alum groups were at about 0.1 to 02%.

[0155] ELISPOT assay was performed with unfractionated PBMC samplescollected two weeks after series of priming injections. The resultsshowed that all Mamu-A*01 vaccinees have developed IFN-g ELISPOTresponse against p11C peptide. These responses in general correlate withtheir tetramer staining, with adenovirus group showing the highestaverage spot counts, followed by the CRL-1005 group, MPL/alum group, andDNA in saline group. The MVA inoculation induced high background spotcounts in all three rhesus monkeys, thus the results could not beinterpreted meaningfully. It is noted that many vaccinees also developedT cell responses against T cell determinants other than p11C epitope, asboth N- and C-terminal peptide pools contained no p11C epitope sequence.This is especially noticeable for the adenovirus-immunized group, as allthree monkeys have developed responses against extra-p11C responses.

[0156] Challenge data and disease progression—Twelve weeks post theboost, all monkeys were challenged with 50 MID50 of SHIV 89.6Pintravenously. All monkeys were infected, demonstrated acute viremiawithin 3 weeks post infection that is typical of this viral stock (seeFIGS. 14A-F). Post challenge longitudinal results for peripheralp11C-specific tetramer staining are presented for each group (FIGS.14A-F), as well as CD4 T cell counts and plasma viral load for eachgroup (FIGS. 15A-L). The six monkeys in the naïve control groupdemonstrated the rapid CD4 T cell lymphopenia within 3 weeks afterchallenge. The peak viral loads reached 10{circumflex over ( )}8copies/ml that is typical for SHIV 89.6P. There is very low tetramerstaining cells in the two Mamu-A*01 monkeys in this group. One exceptionin this group is a Mamu-A*01 monkey, V388, who maintained CD4 countsover 300 cell/ul. Its viral load gradually declined after peaked aroundday 14 to 17 coincide with an about 2% tetramer staining for p11Cepitope. Low but consistent level p11C tetramer staining is detectableduring the course of follow-up, maintained at about 0.8-1.0%. No monkeysin MVA-vaccinated group were protected against CD4 loss, although 15Gmaintained CD4 T cell counts above 300 cells/ml through the acute phase.The peak viremia for the group was indistinguishable for that of naïvecontrol group, but their VL at the chronic stage were significantlylower than those of control group. At the day 180 post challenge, twoout three monkeys maintained viral load around 1000 copies/mL, while thethird on 10000 copies/mL. All three vaccinees in the adenovirus groupachieved protection against CD4 loss upon viral challenge, as all ofthem maintained CD4 counts well above 500 cells/ml though the course ofthe study. It is also noticeable that peak viral loads in all threemonkeys were at least one log lower than those in control naïve monkeys,and viral loads in all three monkeys become undetectable after day 180post challenge (under 500 copies/mL). The nine vaccinees in three DNAvaccinated groups were not protected against acute CD4 lymphopenia postviral challenge, except one monkey in MPL/alum group (058R). This monkeymaintained CD4 counts over 500 cells/μL through acute phase. The peakviral loads of these groups were not significantly different from thatof naïve control monkeys. However, the viral loads on day 180 postchallenge of all three groups were lower than those of naïve controlmonkeys (1-2 log reduction). The most noticeable is the DNA/CRL1005group, where their viral loads of two out of three monkeys were belowthe detection limit (500 copies/mL). Immunodeficiency related diseaseswere reported for five of the six naïve control monkeys, and four havedied or been sacrificed by day 240 post challenge per attendingveterinarians' recommendation. No vaccinees have developed anyimmunodeficiency syndromes or suffered any consistent weight loss.

[0157] Post-challenge Immune response analysis—To define the T cellresponses in control of viral replication post challenge, unfractionatedPBMC were tested in IFN-γ ELISPOT assay against p11C peptide, the SIVgag peptide pool (N-plus C-ter peptide pool), and 89.6P env gp120peptide pool. Strong ELISPOT responses against SIV gag antigen (peptidepool or p11C peptide) were observed in all vaccinees, but none in thenaïve control monkeys. The average of spots for each vaccination groupcorrelates with their p11C tetramer staining. No significant responseswere observed for env 89.6 antigen peptide pool, and in general theywere lower than their respective anti-p11C or anti-SIV gag responses.These results support the immune dominance status of p11C epitope asindicated by longitudinal tetramer staining results. To differentiatethe subset of T cells accounting for IFN-γ production, IFN-γintracellular staining along with T cell surface marker staining (CD3,CD4, CD8) was performed on PBMC cells that were stimulated with thesepeptide pools All vaccinees in adenovirus group exhibited CD4 responseagainst SIV gag, in contrast to naïve control monkeys. No meaningful CD4responses could be registered in the CRL1005 group and the MVA groupbecause of severe CD4 T cell lymphopenia. All monkeys in adenovirusgroup exhibited CD8 T cell responses against SIV gag peptide poolmissing p11C peptide, indicating that additional T cell determinantswere recognized. This is also the case for one monkey in MPL/alum group,058R.

[0158] Serum samples from these monkeys were tested for theirneutralizing titers against SHIV89.6P virus. Neutralizing Abs appearedmostly during week 3-4 post challenge, with some achieved as early asweek 2 post challenge (97×011 and 058R). However, no specificcorrelation with the virological parameters could be identified, as themonkeys in Ad5-SlVgag and DNA/CRL1005 groups controlled their viral loadat the chronic stage with the late appearance of neutralizing Ab titers.These results indicated that cellular immune response induced by SIVgag-based vaccines, not a humoral Ab response, was protective in thisexperiment against challenge of SHIV89.6P. The correlation coefficiencyanalysis was performed on pre challenge tetramer staining result (CMIresponse induced by SIV gag vaccination) and plasma viral load at day180 post challenge. The negative correlation was revealed with R=−0.685and p<0.003.

[0159] Histological analysis was performed on lymph nodes collectedaround day 120 post challenge on selected monkeys. While SHIV RNA couldreadily be detected in the lymph nodes from naïve control monkeys by insitu hybridization, very few infected T cells could be found in theslides derived from LN samples of monkeys in ad5-SIVgag and DNA-CRL1005groups. Quantitation of these SHIV RNA+ cells indicated that there weregreater than 10 fold reduction in number of virus-infected cells in LNfor both Ad5-SIVGag and DNA/CRL1005 groups. Staining CD4 T cells showedthat Ad5-SIVgag vaccinated monkeys preserved the CD4 T cells, while bothnaïve control and DNA/OptiVax groups suffered severe CD4 T celldepletion.

[0160] This example evaluates the role of CD8+ cytotoxic T lymphocytes(CTL) in controlling SIV infection in rhesus macaques. To directlycompare the efficacy of various vaccine vectors for induction ofprotective CMI responses, monkeys were immunized with three vaccinesexpressing SIVmac239 gag: (1) a MVA vector; (2) an adenovirus vector,and (3) a plasmid DNA vector. The DNA vaccine was formulated either insaline, with adjuvants including synthetic polymers (CRL1005, fromCytRx), and monophyspholipid A absorbed on alum (MPL/Alum). Theimmunogenicity of these vaccines was compared directly in rhesus monkeysusing class I tetramer that was specific for a CTL epitope restrictedwith Mamu-A*01 allele. Cellular immune responses were also evaluatedwith peptide pools covering SIV gag region using ELISPOT assay. Threemonths post the last vaccination, all monkeys were challengedintravenously with pathogenic Simian-human Immunodeficiency virus(SHIV), SHIV-89.6P. As shown herein, five out of six naïve controlmonkeys showed rapid loss of CD4⁺ T cells, higher setpoint viral loads,and four succumbed to immunodeficiency syndromes and sacrificed withinsix months post challenge. All the vaccinees were infected butdemonstrated potent amnestic CTL responses judged by SIV gag CTL epitopetetramer staining. All monkeys exhibited various degrees of protectionscorrelated with their pre challenge tetramer staining. Adenoviral vectorvaccinated monkeys were protected from acute CD4⁺ lymphopenia, and theirplasma viral loads were below the detectable limit (<500 copies/mL) sixmonths post challenge. The monkeys in the DNA/CRL1005 group were notprotected from CD4⁺ lymphopenia, but their viral load at 6 months postchallenge were below 500 copies/mL. These results further supportearlier examples disclosed herein: that vaccine-elicited CTL responsesare capable of controlling a highly pathogenic AIDS virus challenge andpreventing immunodeficiency, clinical disease progression, and death.

[0161] In conclusion, the Ad5 and DNA/CRL1005 immunized monkeys hadlower levels of virus in their blood than did the control animals (˜100to 1000-fold lower). Five of 6 controls have exhibited AIDS-relatedillnesses since infection while 0 of 6 adenovirus and DNA/Optivaxvaccinees have shown AIDS-related illnesses. Four out of 6 controls havedied (euthanized at the discretion of attending veterinarians to preventsuffering) while 0 of 15 vaccinees (all groups taken together) havedied. The Adenovirus SIV gag vaccine elicited more potent cellularimmune responses than other vaccines evaluated and apparently protectedmore effectively from SHIV challenge. The degree of protection duringchronic viremia appears to associate with relative immunogenicity priorto challenge, and with the development of multi-epitopic responses afterchallenge. Finally, these data demonstrate that vaccine-mediatedcellular immunity directed only against gag, without priming foranti-env Abs, can provide significant control of SHIV infection inrhesus monkeys

What is claimed is:
 1. A vaccine composition comprising a replicationdefective adenoviral vector comprising at least one gene encoding a HIVgag protein which is codon optimized for expression in a human, and thegene is operably linked to a heterologous promoter and transcriptionterminator.
 2. An adenoviral vaccine vector comprising: a) a replicationdefective adenoviral vector, wherein the adenoviral vector does not havea functional E1 gene, and further comprising: b) a gene expressioncassette comprising: i) a nucleic acid encoding a gag protein which iscodon optimized for expression in a human host; ii) a heterologouspromoter operatively linked to the nucleic acid encoding the gagprotein; and iii) a transcription terminator.
 3. A vector according toclaim 2, wherein the E1 gene has been deleted from the adenoviralvector.
 4. A vector according to claim 3, wherein the gene expressioncassette has replaced the deleted E1 gene.
 5. A vector according toclaim 3, wherein the adenovirus vector does not have a functional E3gene.
 6. A vector according to claim 5 wherein the E3 gene has beendeleted from the replication defective adenoviral vector.
 7. A vectoraccording to claim 6 comprising adenoviral 5 sequences deleted of E1region base pairs (bp) 342-3523 and deleted of E3 region bp28,133-30,818.
 8. A vector according to claim 6 comprising adenoviral 2deleted of E1 region bp 559-3503 and E3 region bp 28,812-29,773.
 9. Avector according to claim 8 comprising the sequence given in FIG.
 6. 10.A vector according to claim 8 wherein the sequence is tPA-gag.
 11. Avector according to claim 2 further comprising a physiologicallyacceptable carrier.
 12. An adenoviral vaccine composition for producingan immune response against human immunodeficiency virus (HIV) in a humancomprising: a) adenovirus serotype 5 sequences bp 1 to bp 341 and bp3534 to 5798; and b) a gene expression cassette, located 3′to adenovirussequence bp 341, comprising: i) a nucleic acid encoding gag which iscodon-optimized and optionally has the tPA leader sequence at its 5′end;ii) a human CMV promoter plus intron A operatively linked to the nucleicacid encoding gag; and iii) a bovine growth hormone transcriptionterminator.
 13. A plasmid vector comprising: a) an adenoviral portioncomprising an adenoviral vector according to claim 2; and b) a plasmidportion.
 14. A cell comprising an adenoviral vector of claim
 2. 15. Amethod of producing the vector of claim 2 comprising introducing theadenoviral vector of claim 2 into a host cell which expresses adenoviralE1 protein, and harvesting the resultant adenoviral vectors.
 16. Amethod according to claim 15 wherein the cell is a 293 cell or PER.C6cell.
 17. A method of generating a cellular immune response against anHIV protein in an individual comprising administering to the individualat least one adenovirus vaccine vector and a vaccine plasmid, whereinsaid adenovirus vaccine vector comprises a) a replication defectiveadenoviral vector, wherein the adenoviral vector does not have afunctional E1 gene, and b) a gene expression cassette comprising: i) anucleic acid encoding gag protein optimized for expression in a humanhost; ii) a heterologous promoter operatively linked to the nucleic acidencoding the gag protein; and iii) a transcription terminator whereinsaid vaccine plasmid comprises: irus portion comprises a) a geneexpression cassette comprising: a nucleic acid encoding a gag protein,wherein the nucleic acid is codon optimized for expression in a humanhost; b) a promoter; and c) a transcription terminator wherein thevaccine plasmid does not contain any adenoviral genes.
 18. A methodaccording to claim 17 comprising administering a vaccine plasmid to theindividual, and after a predetermined minimum amount of time has passed,administering an adenovirus vaccine vector to the individual.
 19. Amethod according to claim 17 comprising administering an adenovirusvaccine vector to the individual, and after a predetermined minimumamount of time has passed, administering a vaccine plasmid to theindividual.
 20. A method according to claim 14 comprising administeringan adenovirus vaccine vector to the individual, and after apredetermined minimum amount of time has passed, re-administering anadenovirus vector to the individual.